One of the most commonly used approaches involving suicide gene transfer/prodrug therapy is the herpes simplex virus thymidine kinase (HSV-TK)/ganciclovir (GCV) system. While this enzyme/prodrug approach has produced tumor regressions in several animal models, results in patients are less encouraging. A major obstacle to clinical efficacy for this and other modes of gene therapy is the low extent of gene transfer in vivo, typically to fewer than 10 percent of cells within a tumor. To improve therapy with this approach, bystander killing must be improved. In the previous application, we demonstrated that HSV-TK/GCV is uniquely able to induce multi-log cell killing in a variety of human tumor cell lines, due to a novel mechanism in which low levels of GCV triphosphate are highly cytotoxic. These studies also showed that bystander killing was effective because transfer of even low levels of GCV phosphates to non-HSV-TK-expressing cells resulted in high cytotoxicity. Further studies demonstrated that a small reduction in the competing dGTP pool by the addition of a ribonucleotide reductase inhibitor, hydroxyurea, enhanced killing of HSVTK cells additively whereas it produced a synergistic increase in bystander cell killing. Preliminary studies suggest that this effect can be achieved in animal models in vivo. In the current application, these studies will be extended by determining whether more potent inhibitors of ribonucleotide reductase, or more specific inhibitors of dGTP synthesis, can improve this bystander cell killing in vitro and in vivo. In addition, we have noted a novel mechanism of GCV phosphate transfer in cells that does not involve GJIC, and we have devised a panel of isogenic cell lines that vary in the level of GJIC to compare the roles of the novel mechanism vs. GJIC in bystander cell killing. The ability of the biochemical modulators to enhance bystander killing in cells either proficient or deficient in GJIC will be evaluated. The efficacy of increased GJIC vs. biochemical modulation to eradicate tumors in animal models in which only a portion of the tumor expresses HSV-TK will be determined. The results from these studies should prove useful in understanding the role of GJIC in bystander killing under clinically relevant conditions, and may help in designing clinical protocols combining HSV-TK/prodrug therapy with a biochemical modulator.